Abstract: Mammalian genomes have a complex physical structure shaped by myriad duplications, deletions and rearrangements, and this structure varies considerably among the populations and individuals of a species. These "structural variations" are of special importance to our understanding of evolution and disease because single mutational events can affect large phenotypic changes, and because mutation rates vary dramatically among different genomic loci. We are only in the very early stages of understanding how structurally plastic genomes truly are, and why they are this way. Massively parallel paired-end DNA sequencing now offers the opportunity, in theory, to reconstruct the architecture of entire genomes on a routine basis. However, the practical utility of these methods remains limited by the significant computational challenges posed by proper data interpretation, and by cost. Over the past year we have developed novel experimental and computational tools, and we are now close to our initial goal of being able to comprehensively map structural variation in mammalian genomes, at reasonable cost and with modest computing power. We propose to apply these tools to examine structural variation in three especially revealing contexts: among diverse mouse strains with shared genealogical origins, among related mouse colonies separated by ~2,000 generations of breeding, and among single cells from diverse somatic lineages of the body and brain. In each case we will systematically identify and characterize "hotspot" loci that mutate at elevated rates. These studies will yield an unbiased evaluation of the extent and origin of structural variation in mammalian genomes, and will enable us pursue our final goal: to develop a high-throughput platform for identifying factors that affect structural mutation rates. This work has immediate relevance to medicine considering that structural genomic variation has emerged as a major cause of both inherited and spontaneous human disease. Public Health Relevance: Structural variation is a ubiquitous feature of mammalian genomes, but little is known about the underlying process through which these duplications, deletions and rearrangements of DNA arise. This question is of great relevance to public health because spontaneous structural mutations in the germline contribute to a number of spontaneous human diseases, including autism and schizophrenia, and because mutations arising in somatic cells can lead to acquired diseases such as cancer. We will use powerful new DNA sequencing technologies and novel computational methods to investigate this process.